In previous years reports we described the identification of a major immunostimulatory molecule in T.gondii that promotes host resistance by stimulating the production of IL-12 by dendritic cells through a Toll-like receptor, TLR11. This parasite molecule is a structural protein, profilin, which is found exclusively in eukaryotes where it is typically involved in actin stabilization. In a collaborative study with the laboratory of Dominque Soldati (University of Geneva) we were able to both examine the role of profilin in the parasite life cycle and assess its requirement in the induction of IL-12 in vivo and in vitro by the live parasite. In this work, the Soldati lab generated parasites that were conditionally knocked out in profilin or in which T.gondii gene was replaced with a gene from malaria (P.falciparum) By this approach it was shown that T.gondii profilin, while not required for intracellular growth, is indispensable for gliding motility, host cell invasion, active egress from host cells, and virulence in mice. Furthermore, parasites lacking profilin were unable to induce TLR11-dependent production of IL-12 in vitro and in vivo. These findings establish profilin as an essential element of two aspects of the T. gondii host-parasite relationship. Thus, in common with bacterial flagellin (which is recognized by TLR-5), profilin plays a role in motility while serving as a microbial ligand that triggers the host innate immune system.[unreadable] In a second major project, we addressed the mechanism by which T.gondii triggers the induction of CD8+ T cells that are required for control of chronic infection. CD8+T cell priming depends on the presentation by dendritic cells (DC) of antigenic peptides loaded onto class I MHC molecules in the cytoplasm of these host cells. Since Toxoplasma gondii resides in a specialized intracellular compartment, the parasitophorous vacuole (PV), it is unclear how T. gondii derived antigens access the MHC class I pathway for presentation to CD8+ T cells. In our work this year we demonstrated a major role for host endoplasmic reticulum (hER)-PV interaction in this process. A direct correlation between hER-PV fusion as evidenced by electron microscopy and T cell priming was observed in actively infected DC. In contrast, no hER association with phagosomes or Ag presentation activity was observed in DC containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite derived Ag was blocked when hER retrotranslocation (a process that moves proteins from the hER to the cytoplasm) was inhibited. These findings demonstrate that pathogen-driven hER-PV fusion can serve as an important route of Ag entry into the MHC class I pathway for vacuole dwelling intracellular microbes such as T. gondii thereby providing an explanation for how such organisms can stimulate strong CD8+ T responses despite their sequestration from the host cytoplasm.